Genetic variation in the most primitive Clivia species

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Date
2010-11
Authors
Van der Westhuizen, Hester Maria
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Publisher
University of the Free State
Abstract
English: The genus Clivia Lindl., which belongs to the family Amaryllidaceae J. St-Hil. (1805), is comprised out of seven different species. Clivia nobilis, C. caulescens, C. miniata, C. gardenii, C. mirabilis, C. robusta and the natural hybrid Clivia xnimbicola all forms part of this genus. Clivia mirabilis is found in the Northern Cape Province and is geographically isolated from the six other species which grows along the Eastern Coast and escarpment of South Africa. Conrad et al. (2003) proved that C. mirabilis and C. nobilis were the two most primitive species in the genus Clivia. During this study sequencing results were used to detect barcodes/SNPs for C. nobilis and C. mirabilis and to reveal genetic variation between the Clivia species. Clivia nobilis and C. mirabilis were tested with cross-species microsatellite makers to reveal intraspecific variation. Seven different gene regions were sequenced. Six were chloroplast regions, namely the atpH-I, matK, rpoB, rpoC1, rpl16, the trnL-F regions and one was a nuclear region, ITS1. The regions used for sequencing were evaluated as potential barcoding/SNP regions for future use. They were also used to infer the evolutionary development of C. nobilis and C. mirabilis. All seven Clivia species were analysed but this study focused mainly on morphologically different specimens of C. nobilis and C. mirabilis. The sequences were aligned and edited with Geneious Pro. A total of forty-seven polymorphic sites were observed between al seven species. Within the rpl16 region eleven parsimony informative sites were observed. The matK and trnL-F regions each had eight parsimony informative sites. ITS1 had three sites and rpoB and rpoC1, one parsimony informative site each. Within the atpHI region no parsimony informative sites were observed. The sequencing data obtained could be used for species identification and, therefore, showed great potential as barcoding regions. We propose that matK, rpl16 and trnL-F are used as a barcode in C. nobilis and C. mirabilis because they had the most parsimony informative sites. The cladogram obtained from the combined data set (atpH-I, rpoB, rpoC1, matK and trnL-F) confirmed that C. nobilis and C. mirabilis are two separate species. Clivia caulescens and C. xnimbicola forms a monophyletic group. Within the rpl16 chloroplast region intraspecific variation in C. mirabilis and interspecific variation between C. nobilis and C. mirabilis were observed. The phylogentic tree representing the sequencing results of the rpl16 region revealed three distinctive groups within the four different C. mirabilis populations. Two plants within one of the Donkerhoek populations showed more variation than the rest of the population. The rpl16 gene region proved to be ideal in order to test intraspecific variation in C. nobilis and C. mirabilis. To evaluate the use of cross-species markers, microsatellite makers designed for Phaedranassa tunguraguae, Hymenocallis coronia and Clivia miniata were tested on C. nobilis and C. mirabilis. Although amplification was obtained, in most cases the results could not be optimized in order to provide reliable analysis. In future species specific primers for C. nobilis and C. mirabilis will be developed. This study undoubtedly identified barcodes/SNPs for C. nobilis and C. mirabilis which can be used to eliminated mistaken identity. Gene regions specific for intra- and interspecific variation were identified and can be used in future for population studies.
Afrikaans: Die genus Clivia Lindl., behoort aan die familie Amaryllidaceae J. St-Hil (1805) en bestaan uit sewe verskillende spesies. Die spesies is C. nobilis, C. caulescens, C. miniata, C. gardenii, C. mirabilis, C. robusta en die natuurlike baster Clivia xnimbicola. Clivia mirabilis word gevind in die Noord Kaap Provinsie en is geografies geïsoleer van die ses ander Clivia spesies wat groei langs die Oos-Kus en platorand van Suid-Afrika. Conrad et al. (2003) het bevind dat C. mirabilis en C. nobilis die twee primitiefste spesies in die genus Clivia is. DNA volgordebepalings is gebruik om strepieskodes vir C. nobilis en C. mirabilis te bepaal en om genetiese variasie tussen die verskillende Clivia spesies te ondersoek. Nie-spesie-spesifieke mikrosatelliet merkers is gebruik om intraspesifieke variasie in C. nobilis en C. mirabilis te ondersoek. DNA volgordes van sewe verskillende geengebiede is bepaal. Ses was chloroplasgebiede, naamlik atpH-I, matK, rpoB, rpoC1, rpl16, trnL-F streke en een kerngebied, naamlik ITS1. Die DNA volgordebepalings is ook geëvalueer as potensiële toekomstige strepieskode/SNP gebiede. Al sewe Clivia spesies is geanaliseer, maar die studie het gefokus op morfologiese verskillende eksemplare van C. nobilis en C. mirabilis. Die volgordes is saamgevoeg en gewysig met Geneious Pro. ʼn Totaal van sewe-en-veertig verskillende polimorfiese posisies is in die sewe spesies waargeneem. Binne die rpl16 gebied is elf parsinomies informatiewe posisies waargeneem. Die matK en trnL-F streke het elk agt informatiewe posisies getoon. ITS1 het drie posisies en rpoB en rpoC1, elk een parsinomies informatiewe posisie. Binne die atpH-I gebied was daar geen parsinomies informatiewe posisies nie. Die DNA volgordebepalings kan dus gebruik word om spesies te identifiseer, en is daarom potensiële strepieskode gebiede. Ons stel voor dat ʼn kombinasie van matK, rpl16 en trnL-F gebruik word as ʼn strepieskode vir C. nobilis en C. mirabilis, weens hul hoë aantal parsinomies informatiewe posisies. Die kladogram wat deur die saamgestelde datastel (atpH-I, rpoB, rpoC1, matK and trnL-F) verkry is, het bevestig dat C. nobilis en C. mirabilis twee verskillende spesies is. Clivia caulescens en C. xnimbicola vorm ʼn monofiletiese groep. Alhoewel C. nobilis en C. mirabilis merkbaar verskil van die ander Clivia spesies, verskil hul steeds van mekaar. Die rpl16 chloroplasgebied toon intraspesifieke variasie aan in C. mirabilis en interspesifieke variasie tussen C. nobilis en C. mirabilis. Die filogram gebaseer op volgordebepalings van die rpl16 gebied, toon aan dat binne die vier C. mirabilis populasies drie kenmerkende groepe geïdentifiseer kan word. Twee plante binne een van die Donkerhoek populasies vertoon meer variasie as die res van die populasie. Die rpl16 geen gebied blyk die beste te wees om intraspesifieke variasie tussen C. nobilis and C. mirabilis aan te toon. Om die gebruik van kruis spesies mikrosatelliet merkers te evalueer is merkers gebruik wat oorspronklik ontwerp is vir Phaedranassa tunguraguae, Hymenocallis coronia en Clivia miniata. Alhoewel amplifisering verkry is, kon die resultate meestal nie genoegsaam geoptimaliseer word om betroubare analise te verseker nie. In die toekoms sal die ontwikkeling van spesies spesifieke inleiers vir C. nobilis en C. mirabilis noodsaaklik wees. Die studie het ongetwyfeld strepieskodes/SNPs geïdentifiseer vir C. nobilis en C. mirabilis wat gebruik kan word om verkeerdelike identifisering te elimineer. Geenstreke, spesifiek vir intra- en interspesifieke variasie, is geïdentifiseer en kan in die toekoms gebruik word vir populasie studies.
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Keywords
Dissertation (M.Sc. (Genetics))--University of the Free State, 2010, Clivia -- Classification, Clivia -- South Africa -- Genetics, Hybridization, Amaryllidaceae, Phylogenetic relationship, trnL-F, rpl16, rpoC1, rpoB, matK, atpH-I, Barcoding, Microsatellite markers
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